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A Two-Step Pretargeted Nanotherapy for CD20 Crosslinking May Achieve Superior Anti-Lymphoma Efficacy to Rituximab.

Chu TW, Zhang R, Yang J, Chao MP, Shami PJ, Kopeček J - Theranostics (2015)

Bottom Line: Consecutive treatment with the two components resulted in CD20 clustering on the cell surface and effectively killed malignant B-cells in vivo.In a mouse model of human non-Hodgkin lymphoma (NHL), increasing the time lag from 1 h to 5 h resulted in dramatically improved tumor growth inhibition and animal survival.In summary, our approach may constitute a novel treatment for NHL and other B-cell malignancies with significant advantages over conventional chemo-immunotherapy.

View Article: PubMed Central - PubMed

Affiliation: 1. Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, UT 84112, USA.

ABSTRACT
The use of rituximab, an anti-CD20 mAb, in combination with chemotherapy is the current standard for the treatment of B-cell lymphomas. However, because of a significant number of treatment failures, there is a demand for new, improved therapeutics. Here, we designed a nanomedicine that crosslinks CD20 and directly induces apoptosis of B-cells without the need for toxins or immune effector functions. The therapeutic system comprises a pretargeting component (anti-CD20 Fab' conjugated with an oligonucleotide1) and a crosslinking component (N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer grafted with multiple complementary oligonucleotide2). Consecutive treatment with the two components resulted in CD20 clustering on the cell surface and effectively killed malignant B-cells in vivo. To enhance therapeutic efficacy, a two-step pretargeting approach was employed. We showed that the time lag between the two doses can be optimized based on pharmacokinetics and biodistribution of the Fab'-oligonucleotide1 conjugate. In a mouse model of human non-Hodgkin lymphoma (NHL), increasing the time lag from 1 h to 5 h resulted in dramatically improved tumor growth inhibition and animal survival. When the 5 h interval was used, the nanotherapy was more efficacious than rituximab and led to complete eradication of lymphoma cells with no signs of metastasis or disease recurrence. We further evaluated the nanomedicine using patient mantle cell lymphoma cells; the treatment demonstrated more potent apoptosis-inducing activity than rituximab hyper-crosslinked with secondary antibodies. In summary, our approach may constitute a novel treatment for NHL and other B-cell malignancies with significant advantages over conventional chemo-immunotherapy.

No MeSH data available.


Related in: MedlinePlus

Eradication of lymphoma B-cells by drug-free macromolecular therapeutics. Mice i.v. injected with Raji-luc cells were exposed to different treatments as indicated in Fig. 4. (A) Representative ex vivo bioluminescent images of the control mice (PBS) and the mice treated consecutively with Fab'-MORF1 and P-MORF2 using 5 h as an interval (Cons 5h). In vivo images of the same mice are shown alongside. bra: brain, hea: heart, lun: lung, liv: liver, spl: spleen, kid: kidney, s.c.: spinal cord, l.n.: lymph node, tib: tibia, fem: femur, mus: muscle, sto: stomach, int: intestine. (B) Flow cytometry analysis of residual Raji-luc cells in the bone marrow (BM) of mice. BM cells isolated from mouse femur and Raji-luc cells (with GFP expression) from culture flasks were stained with an APC-labeled mouse anti-human CD19 antibody. (C) Comparison of % lymphoma cells in the bone marrow of control mice (PBS) and the nanomedicine-treated mice (Cons 1h and Cons 5h) as analyzed by flow cytometry. Each data point represents an individual mouse (n = 6 per group); mean % is indicated. Statistics was performed with Student's t test of unpaired samples (*: p < 0.05, **: p < 0.005).
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Figure 6: Eradication of lymphoma B-cells by drug-free macromolecular therapeutics. Mice i.v. injected with Raji-luc cells were exposed to different treatments as indicated in Fig. 4. (A) Representative ex vivo bioluminescent images of the control mice (PBS) and the mice treated consecutively with Fab'-MORF1 and P-MORF2 using 5 h as an interval (Cons 5h). In vivo images of the same mice are shown alongside. bra: brain, hea: heart, lun: lung, liv: liver, spl: spleen, kid: kidney, s.c.: spinal cord, l.n.: lymph node, tib: tibia, fem: femur, mus: muscle, sto: stomach, int: intestine. (B) Flow cytometry analysis of residual Raji-luc cells in the bone marrow (BM) of mice. BM cells isolated from mouse femur and Raji-luc cells (with GFP expression) from culture flasks were stained with an APC-labeled mouse anti-human CD19 antibody. (C) Comparison of % lymphoma cells in the bone marrow of control mice (PBS) and the nanomedicine-treated mice (Cons 1h and Cons 5h) as analyzed by flow cytometry. Each data point represents an individual mouse (n = 6 per group); mean % is indicated. Statistics was performed with Student's t test of unpaired samples (*: p < 0.05, **: p < 0.005).

Mentions: Ex vivo luciferase imaging was performed to examine tumors in multiple organs and tissues (Fig. 6A). Results were concordant with in vivo imaging and showed that Raji-luc lymphoma cells readily infiltrated in the femora, tibiae and lymph nodes of the negative control mice (PBS). Extensive infiltration of the spinal cord was also seen, likely accounting for the occurrence of hind-limb paralysis 28. In some cases, we observed light to moderate tumor signals in the lung, liver or brain (Supplementary Material: Fig. S7) of control animals. However, most of the surviving mice (5 out 6) that were treated with the nanomedicine using a 5 h interval (Cons 5h) were completely tumor-free. Furthermore, we used flow cytometry to quantitatively analyze residual lymphoma B-cells in the femoral bone marrow (BM) of mice (Fig. 6B and 6C). Raji-luc cells harbor a dual reporter expressing both luciferase and green fluorescence protein (GFP) 22. We additionally stained the cells with an anti-human CD19 antibody 24 and analyzed the percentage of GFP+ CD19+ cells (Supplementary Material: Fig. S8). Results showed that the negative control mice (PBS) harbored substantial amounts of Raji-luc cells in the bone marrow (average 6.4%). The mice treated with the nanomedicine using a 1 h interval (Cons 1h) demonstrated significant improvement (average 2.7%). In the group that was treated using optimized treatment conditions (Cons 5h), all mice, including five long-term survivors and one animal sacrificed on day 105 due to a large abdominal tumor, had 0% lymphoma B-cells in the bone marrow. Histology further confirmed that the long-term surviving mice were tumor-free (Supplementary Material: Fig. S9). Pathological examination suggested no acute or chronic toxicity caused by drug-free macromolecular therapeutics in any of the tissues evaluated, which corresponded to a stable body weight (Supplementary Material: Fig. S10). These results demonstrate excellent anti-NHL efficacy of the nanotherapeutics with the 2 components (Fab'-MORF1 and P-MORF2) administered at an optimal interval of 5 h. A low dose (58 μg × 3) of the pretargeting agent with a 5× excess of effectors was able to completely eradicate lymphoma B-cells in mice and was not toxic to normal tissues.


A Two-Step Pretargeted Nanotherapy for CD20 Crosslinking May Achieve Superior Anti-Lymphoma Efficacy to Rituximab.

Chu TW, Zhang R, Yang J, Chao MP, Shami PJ, Kopeček J - Theranostics (2015)

Eradication of lymphoma B-cells by drug-free macromolecular therapeutics. Mice i.v. injected with Raji-luc cells were exposed to different treatments as indicated in Fig. 4. (A) Representative ex vivo bioluminescent images of the control mice (PBS) and the mice treated consecutively with Fab'-MORF1 and P-MORF2 using 5 h as an interval (Cons 5h). In vivo images of the same mice are shown alongside. bra: brain, hea: heart, lun: lung, liv: liver, spl: spleen, kid: kidney, s.c.: spinal cord, l.n.: lymph node, tib: tibia, fem: femur, mus: muscle, sto: stomach, int: intestine. (B) Flow cytometry analysis of residual Raji-luc cells in the bone marrow (BM) of mice. BM cells isolated from mouse femur and Raji-luc cells (with GFP expression) from culture flasks were stained with an APC-labeled mouse anti-human CD19 antibody. (C) Comparison of % lymphoma cells in the bone marrow of control mice (PBS) and the nanomedicine-treated mice (Cons 1h and Cons 5h) as analyzed by flow cytometry. Each data point represents an individual mouse (n = 6 per group); mean % is indicated. Statistics was performed with Student's t test of unpaired samples (*: p < 0.05, **: p < 0.005).
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Figure 6: Eradication of lymphoma B-cells by drug-free macromolecular therapeutics. Mice i.v. injected with Raji-luc cells were exposed to different treatments as indicated in Fig. 4. (A) Representative ex vivo bioluminescent images of the control mice (PBS) and the mice treated consecutively with Fab'-MORF1 and P-MORF2 using 5 h as an interval (Cons 5h). In vivo images of the same mice are shown alongside. bra: brain, hea: heart, lun: lung, liv: liver, spl: spleen, kid: kidney, s.c.: spinal cord, l.n.: lymph node, tib: tibia, fem: femur, mus: muscle, sto: stomach, int: intestine. (B) Flow cytometry analysis of residual Raji-luc cells in the bone marrow (BM) of mice. BM cells isolated from mouse femur and Raji-luc cells (with GFP expression) from culture flasks were stained with an APC-labeled mouse anti-human CD19 antibody. (C) Comparison of % lymphoma cells in the bone marrow of control mice (PBS) and the nanomedicine-treated mice (Cons 1h and Cons 5h) as analyzed by flow cytometry. Each data point represents an individual mouse (n = 6 per group); mean % is indicated. Statistics was performed with Student's t test of unpaired samples (*: p < 0.05, **: p < 0.005).
Mentions: Ex vivo luciferase imaging was performed to examine tumors in multiple organs and tissues (Fig. 6A). Results were concordant with in vivo imaging and showed that Raji-luc lymphoma cells readily infiltrated in the femora, tibiae and lymph nodes of the negative control mice (PBS). Extensive infiltration of the spinal cord was also seen, likely accounting for the occurrence of hind-limb paralysis 28. In some cases, we observed light to moderate tumor signals in the lung, liver or brain (Supplementary Material: Fig. S7) of control animals. However, most of the surviving mice (5 out 6) that were treated with the nanomedicine using a 5 h interval (Cons 5h) were completely tumor-free. Furthermore, we used flow cytometry to quantitatively analyze residual lymphoma B-cells in the femoral bone marrow (BM) of mice (Fig. 6B and 6C). Raji-luc cells harbor a dual reporter expressing both luciferase and green fluorescence protein (GFP) 22. We additionally stained the cells with an anti-human CD19 antibody 24 and analyzed the percentage of GFP+ CD19+ cells (Supplementary Material: Fig. S8). Results showed that the negative control mice (PBS) harbored substantial amounts of Raji-luc cells in the bone marrow (average 6.4%). The mice treated with the nanomedicine using a 1 h interval (Cons 1h) demonstrated significant improvement (average 2.7%). In the group that was treated using optimized treatment conditions (Cons 5h), all mice, including five long-term survivors and one animal sacrificed on day 105 due to a large abdominal tumor, had 0% lymphoma B-cells in the bone marrow. Histology further confirmed that the long-term surviving mice were tumor-free (Supplementary Material: Fig. S9). Pathological examination suggested no acute or chronic toxicity caused by drug-free macromolecular therapeutics in any of the tissues evaluated, which corresponded to a stable body weight (Supplementary Material: Fig. S10). These results demonstrate excellent anti-NHL efficacy of the nanotherapeutics with the 2 components (Fab'-MORF1 and P-MORF2) administered at an optimal interval of 5 h. A low dose (58 μg × 3) of the pretargeting agent with a 5× excess of effectors was able to completely eradicate lymphoma B-cells in mice and was not toxic to normal tissues.

Bottom Line: Consecutive treatment with the two components resulted in CD20 clustering on the cell surface and effectively killed malignant B-cells in vivo.In a mouse model of human non-Hodgkin lymphoma (NHL), increasing the time lag from 1 h to 5 h resulted in dramatically improved tumor growth inhibition and animal survival.In summary, our approach may constitute a novel treatment for NHL and other B-cell malignancies with significant advantages over conventional chemo-immunotherapy.

View Article: PubMed Central - PubMed

Affiliation: 1. Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, UT 84112, USA.

ABSTRACT
The use of rituximab, an anti-CD20 mAb, in combination with chemotherapy is the current standard for the treatment of B-cell lymphomas. However, because of a significant number of treatment failures, there is a demand for new, improved therapeutics. Here, we designed a nanomedicine that crosslinks CD20 and directly induces apoptosis of B-cells without the need for toxins or immune effector functions. The therapeutic system comprises a pretargeting component (anti-CD20 Fab' conjugated with an oligonucleotide1) and a crosslinking component (N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer grafted with multiple complementary oligonucleotide2). Consecutive treatment with the two components resulted in CD20 clustering on the cell surface and effectively killed malignant B-cells in vivo. To enhance therapeutic efficacy, a two-step pretargeting approach was employed. We showed that the time lag between the two doses can be optimized based on pharmacokinetics and biodistribution of the Fab'-oligonucleotide1 conjugate. In a mouse model of human non-Hodgkin lymphoma (NHL), increasing the time lag from 1 h to 5 h resulted in dramatically improved tumor growth inhibition and animal survival. When the 5 h interval was used, the nanotherapy was more efficacious than rituximab and led to complete eradication of lymphoma cells with no signs of metastasis or disease recurrence. We further evaluated the nanomedicine using patient mantle cell lymphoma cells; the treatment demonstrated more potent apoptosis-inducing activity than rituximab hyper-crosslinked with secondary antibodies. In summary, our approach may constitute a novel treatment for NHL and other B-cell malignancies with significant advantages over conventional chemo-immunotherapy.

No MeSH data available.


Related in: MedlinePlus